Next generation sequencing (NGS) libraries are collections of DNA fragments whose nucleotide sequences will be determined. The sources of DNA for insertion into these libraries are typically genomic DNA that has been fragmented to a desired length, or copies of the transcriptome from a given cell population. Transcriptome libraries are generated by making a cDNA copy of an RNA population, creating a complement to each DNA strand, thereby generating double-stranded DNA, and then ligating the double-stranded DNAs to library-specific adaptors. The cDNA can be synthesized by using random primers, sequence-specific primers or primers containing oligo dT tails to prime a population of transcripts that are polyadenylated. Frequently, these fragment populations contain DNA that is not of interest to a particular study, and in some cases, these non-desired DNA sequences represent a very significant percentage of the overall DNA population. For example, in whole transcriptome studies, ribosomal RNA (rRNA) sequences comprise the majority (60-90%) of all fragments in a typical cDNA library, absent steps to remove rRNA from the samples. In another example, gene expression profiling from peripheral blood is primarily concerned with mRNA from peripheral blood mononuclear cells (PBMCs), which make up less than 0.1% of the whole blood sample. Reduction of globin RNA from red blood cells, which make up majority of the cells in the blood sample, is desirable in such assays.
In the case of rRNA removal or depletion, three general methods have been described: 1) removal of rRNA from the starting population; 2) differential priming using oligo dT primers (i.e. priming polyadenylated transcripts only); and 3) differential priming where primers complementary to rRNA sequences are specifically eliminated (or under-represented) in a primer pool (Not-So-Random or NSR primer approach; see Armour et al., 2009). Priming a total RNA population with primers that only recognize poly(A)-sequences is problematic for two reasons. First, it cannot be used with prokaryotic organisms because prokaryotic mRNAs do not contain poly(A)-sequences at their 3′ ends. Second, even with eukaryotic RNA samples, many biologically important elements, such as regulatory transcripts, are not polyadenylated and are therefore lost from the library with oligo dT priming. While NSR priming strategies can be effective when designed to specific organisms, NSR priming can cause distortions in the sample populations when a less optimized set of primers is employed across a broader range of sample types.
There is a need for improved methods for removal of specific non-desired DNA fragments from NGS libraries. Such methods would ideally enable starting with an unbiased template population and eliminating non-desired DNA fragments in a sequence-specific manner after the NGS library has been generated. The invention described herein fulfills this need.